Erythropoietin (EPO), a glycoprotein having the molecular weight of 30,000 to 34,000, stimulates production of a red blood cell. This protein starts its functions by binding with receptors on erythrocyte precursor cells to result in increase of calcium ion concentration in a cell, increase of biosynthesis of DNA, and stimulation for the formation of hemoglobin and the like. Recombinant human EPO (rhEPO) has been used for the treatment of anemia from renal failure, anemia of a premature baby, anemia from hypothyroidism, anemia from malnutrition, and the like. On the other hand, a clinical use of rhEPO is on the increase. However, the use of rhEPO may cause inconvenience and high costs from the administration of on average 3 times a week due to the short half-life of the rhEPO. Thus, if the in vivo activity of EPO is maintained for a long time, the administration frequency of EPO may be decreased very much.
In vivo activity of EPO is proportional to the in vivo half-life of EPO. It is known that in vivo half-life of EPO is correlated to the content of sialic acid that is located at the terminal of carbohydrate chains of EPO. Therefore, in vivo activity of EPO is highly dependent on the content of carbohydrate chains. On the other hand, the forms of carbohydrate are different according to the kinds of the cells in which EPO is expressed, and therefore, the same glycoproteins may show different carbohydrates if they are expressed in different cells. Some bacteria, for example E. coli, are known not to be capable of attaching the carbohydrate chains to the protein. Generally, proteins expressed in E. coli do not contain the carbohydrate chains, and thus, E. coli-derived EPO, which does not contain the carbohydrate chains, exhibits both enhanced in vitro activity and decreased in vivo activity. Deglycosylated EPO is rapidly eliminated from the human body and has an extremely short half-life. In conclusion, the carbohydrate chains play a very important role in the activity of EPO.
Many studies have been conducted to enhance the activity of EPO. The main method is substitution of some amino acids of EPO by mutagenesis. For example, PCT/US94/09257 filed by Amgen, and titled “Erythropoietin Analog”, discloses a method to increase the half-life of EPO by increasing the carbohydrate contents using mutagenesis. Also, an attempt was made to increase the half-life of EPO by formation of EPO dimer. See, A. J. Sytkowski et al., J.B.C. vol. 274, No. 35, pp 24773-24778. Besides, another known method is to enhance in vivo activity of EPO by fusing new amino acids, peptides or protein fragment with EPO and increasing the carbohydrate content, i.e., sialic acid content of EPO. However, all amino acids, peptides or heterogeneous protein fragments may not be used in such methods. In most cases, such fusions result in decrease or loss of inherent activity of protein and may cause a problem of antigenicity when used in vivo.
Fusion proteins or chimeric proteins, etc. have been studied, for example, for follicle stimulating hormone, a sex hormone. See, Furuhashi et al., 1995, Mol. Endocrinol.). However, the methods have not been applied to industry since protein modification itself have many risks, and the target protein is not readily obtained without professional skills, and the inherent activity of protein may be decreased or lost to cause the opposite result.